Enzyme sensors are sensors where a chemical species to be measured (an analyte) undergoes an enzyme catalysed reaction in the sensor before detection. The reaction between the analyte and the enzyme (for which the analyte is a substrate), or a cascade of enzymes, yields a secondary species which concentration (under ideal conditions) is proportional with or identical to the concentration of the analyte. The concentration of the secondary species is then detected by a transducer, e.g., by means of an electrode.
The enzyme of an enzyme sensor is typically included in a sensor membrane suitable for contacting a fluid sample. Most typically, the enzyme is included in a separate enzyme layer of the sensor membrane, which is separated from the fluid sample by means of a cover membrane. Hence, the analyte is contacted with the enzyme after diffusion through the cover membrane of the sensor, the enzyme/analyte reaction then takes place, and the secondary species then diffuses to the detector part of the sensor, e.g., an electrode, to yield a response related to the analyte concentration.
In an enzyme sensor, the cover membrane should, on the one hand, have a suitable porosity so that the analyte diffuses from the fluid sample to the enzyme layer in a controlled manner, i.e., the diffusion resistance for the analyte should preferably be so that the conversion of the analyte to the secondary species is only limited by the analyte concentration.
On the other hand, the cover membrane should be impermeable or substantially impermeable to proteins on either side of the cover membrane and particularly to the enzyme of the enzyme layer in order to avoid leaching of the enzyme into the fluid sample.
Porous cover membranes useful for enzyme sensors include track-etched membranes (i.e., membranes where discrete, through-going pores are created by atom bombardment followed by etching) and solvent-cast membranes having tortuous pores (i.e., pores formed by connected pore cells). The latter may be prepared by casting the material dissolved in a solvent as a discrete film or in-situ, where the material may comprise a compound that can easily be washed out of the bulk whereby pores are formed.
A conventional, enzyme-based sensor with a track-etched cover membrane has several problems relating to the fact that large molecules (i.e., a molecular weight larger than approximately 1,000 Daltons) are able to diffuse through the pores thereof.
FIGS. 4A and 4B illustrate the problems of cover membranes containing large, track-etched pores for enzyme-based sensors. It can be seen how the enzyme (25) can migrate into the pores of the porous cover membrane (26). The varying degree of filling (27) leads to variations in sensitivity. Sensor linearity can be compromised, e.g., by enzymes penetrating to the outside of the cover membrane (28), or because the analyte in high and low level samples is being degraded by two different enzyme populations (active enzyme at low analyte levels (33) and active enzyme at high analyte levels (32)). From the sample side, blood proteins (31) can enter the pores and precipitate on the pore walls and thereby decrease the diffusion coefficient of the analyte, thus decreasing the sensor sensitivity when measuring blood samples. Moreover, the mere presence of blood proteins in the pores may give rise to blood bias. A further problem relates to the distribution of the analyte in the enzyme layer, especially where track-etched membranes are used.
If the active enzyme from an underlying enzyme layer is able to migrate into or through the porous cover membrane, it may cause different problems. First of all, a loss of enzyme through the cover membrane (29) causes a reduced lifetime of the sensor unit. Secondly, if the enzyme migrates into the pores of the membrane (27), it will cause varying sensor-to-sensor sensitivity. Furthermore, the sensitivity of the sensor also tends to vary over the first few days until the precipitation/dissolution has come to equilibrium. Such changes are unwanted because they cause variations in sensor performance. If the enzyme precipitates on the outside of the membrane (28), the enzyme will be able to carry out its enzymatic action in the fluid sample. However, in such instances, the amount of analyte (e.g., glucose, lactate, creatine, creatinine) being converted to the secondary species (e.g., H2O2) is not linearly correlated to the concentration of primary analyte, since other factors such as Km and the concentration of other possible substrates (e.g., O2) also influence the conversion rate.
If proteins (e.g., blood proteins) from the fluid sample are able to pass into the pores (31) of the cover membrane, the proteins will remain in the liquid column of the pores and thus lower the area and increase the diffusion length for the analyte into the enzyme layer. Therefore, the sensor will have varying sensitivities for the analyte of fluid samples with varying protein content. This phenomenon is referred to as blood bias which is caused by the blood components occupying the pores of the cover membrane, thereby providing a higher (however varying) effective diffusion resistance in the pores of the cover membrane, and, thus, of the enzyme membrane as such.
Moreover, if the surface of the pores is not totally blood compatible, then the proteins may precipitate on the membrane surface including inside the pores (30) and cause a gradually smaller area (gradually reduced effective pore size) for the analyte to diffuse through into the enzyme layer. This phenomenon is referred to as blood drift which is caused by blood components (in particular, proteins and lipids) precipitating on the inner surface of the pores, and, thus, decreasing sensitivity.
Both these effects (blood bias and blood drift) are especially undesirable for sensors in blood analysers because the known enzyme sensors often are calibrated with aqueous liquids, whereas they are meant to measure blood samples.
Conventional solvent-cast membranes also cause problems of the type described above, such as sensor-variability, linearity, unsatisfactory analyte distribution in the enzyme layer, etc.
Moreover, as it is desirable to use effective rinsing solutions for enzyme sensors, in particular rinsing solutions comprising proteases (e.g., subtilisin). It is also important that such proteases are not able to penetrate into the enzyme layer.
U.S. Pat. No. 4,919,767 discloses an enzyme sensor comprising an enzyme layer and a liquid membrane of a porous material filled with liquid having the ability to let the analyte pass while rejecting other species of the samples.
U.S. Pat. No. 6,413,393 discloses a sensor comprising at least one functional coating layer that includes a UV-absorbing polymer, e.g., a polyurethane, a polyurea or a polyurethane/polyurea copolymer including variants comprising hydrophilic segments such as poly(alkylene glycol) and poly(alkylene oxide). The sensor may comprise an enzyme layer and two functional layers, e.g., an analyte limiting layer and a biocompatible layer.
US Published Application 2003/0217966 A1 discloses an implantable membrane for regulating the transport of analytes therethrough that includes a polyurethane (in particular, a polyether urethaneurea) matrix having a network of microdomains of another polymer which may be of the same type, i.e., a polyether urethaneurea.
U.S. Pat. No. 6,652,720 B1 discloses an electrochemical sensor having at least one electrode and a composite membrane. The composite membrane comprises a diffusion-controlling outer layer comprising a polyurethane-based compound, e.g., a mixture of polyurethanes with different water-uptake properties.
US Published Application 2002/065332 A1 discloses a polymeric reference electrode membrane comprising a porous polymer or a hydrophilic plasticizer in combination with a lipophilic polymer e.g., a polyurethane.
U.S. Pat. No. 6,350,524 B1 discloses a solid-state membrane for a chloride-selective electrode comprising an insoluble metal salt layer and a protecting membrane formed of hydrophilic polyurethane.
U.S. Pat. No. 6,200,772 B1 discloses a sensor device having a membrane comprising a polyurethane modified with a non-ionic surfactant, e.g., an aliphatic polyether.
U.S. Pat. No. 5,322,063 A discloses a homogeneous membrane of a hydrophilic polyurethane composition. The membrane is useful for glucose enzyme sensors.
WO 2003/076648 A1 discloses a planar, thick-film biosensor having a homogeneous layer of a polymer (e.g., an aliphatic polyether urethane) comprising an enzyme and a mediator.
US Published Application 2004/0154933 A1 discloses a polymeric membrane for use in electrochemical sensors. The membrane contains carboxylated polyvinyl chloride, e.g., mixed with a polyurethane.
WO 2004/062020 A2 discloses a gas diffusion layer of a porous polymeric material, e.g., a foam, for a fuel cell. The material may be a polyurethane foam or polyether polyurethane foam. Similarly, US 2004/0001993 A1 discloses a gas diffusion layer for fuel cells.
EP 1 486 778 A2 discloses an electrochemical biosensor comprising a membrane of a polymer having a bio-active agent, e.g., an enzyme, entrapped therein.
WO 92/04438 A1 discloses an electrochemical biosensor having a substrate-limiting layer of a hydrophobic plastic layer, e.g., a polyurethane layer.
EP 0 025 110 A2 discloses an electrochemical sensor having an asymmetric semipermeable membrane of, e.g., polyvinyl chloride.
US Published Application 2004/0011671 A1 discloses an implantable device having an enzyme layer, a bioprotective membrane e.g., a polyurethane layer, and an outer angiogenic layer, e.g., of PTFE, PVF, cellulose esters, PVC, polypropylene, polysulfones or poly(methyl methacrylate).
WO 90/05910 A1 discloses a wholly microfabricated biosensor comprising an enzyme layer and an “analyte attenuation layer”, e.g., a polyurethane layer.
WO 96/26668 A1 discloses an implantable sensor system comprising a flexible capillary membrane which may be coated with a polyurethane or silicone, wherein said capillary membrane is connected to a channel in contact with an enzyme sensor e.g., a glucose sensor.
U.S. Pat. No. 5,523,118 discloses a microporous membrane for transdermal drug delivery patches. The microporous membrane may be made from, e.g., polyvinyl chloride and is coated with a substantially porous coating formed from an adhesive-compatible urethane-based polymer, e.g., an aliphatic polyether urethane dispersion. The urethane-based polymer coats the surface of the microporous membrane, but does not block the pores thereof.
U.S. Pat. No. 6,509,148 discloses biosensors utilizing a hydrophilic polyurethane mixed with an enzyme.
JP 2655727 B2 discloses an enzyme sensor having a substrate limiting layer of polyurethane or cellulose acetate, or a dual layer of these, and an outer biological-body compatible film of polyvinyl alcohol (PVOH).
In view of the above, there is still a need for improved enzyme sensors having cover membranes providing an efficient barrier for enzymes and other proteins and secondary analytes and yet providing excellent and stable diffusion control of the primary analyte to the enzyme layer.